EP2471929A1

EP2471929A1 - Production of high mannose glycosylated proteins stored in the plastid of microalgae

Info

Publication number: EP2471929A1
Application number: EP10016162A
Authority: EP
Inventors: Aude Carlier; Rémy Michel; Nathalie Dufourmantel; Jean-Paul Cadoret; Alexandre Lejeune
Original assignee: ALGENICS
Current assignee: ALGENICS
Priority date: 2010-12-29
Filing date: 2010-12-29
Publication date: 2012-07-04
Also published as: US20140066606A1; EP2658980A1; WO2012089342A1

Abstract

The present invention concerns a transformed microalga producing a protein harboring a non-immunogenic "high mannose" pattern of glycosylation in the plastid of said transformed microalga, wherein 1) said transformed microalga has a Chloroplast Endoplasmic Reticulum (CER); 2) said microalga has been transformed with a nucleic acid sequence operatively linked to a promoter, said nucleic acid sequence encoding an amino acid sequence comprising (i) an amino-terminal bipartite topogenic signal (BTS) sequence composed of at least a signal peptide followed by a transit peptide ; and (ii) The sequence of said protein,3) the xylosyltransferases and fucosyltransferases of said microalga have not been inactivated; 4) the N-acetylglycosyltransferase I of said microalga has not been inactivated, preferably the N-acetylglycosyltranferases II, III, IV, V and VI, mannosidase II and glycosyltransferases of said microalga have not been inactivated

Description

FIELD OF THE INVENTION

The present invention is directed to the use of a transformed microalga for the production of glycosylated proteins harboring a pattern of N-glycosylation with high mannose residues, said glycosylated proteins being targeted and stored in the plastid of said microalga. This invention also encompasses methods of producing said glycosylated proteins.

BACKGROUND OF THE INVENTION

The increasing demand for recombinant drugs has strongly driven the development of various cellular models used as biomanufacturing platforms. Expression systems based on eukaryotic cells are preferentially used for the production of recombinant proteins requiring post-translational modifications (PTM) for their biological activity and/or stability. N-glycosylation is a major PTM characterized by the attachment of glycans onto certain asparagine residues of proteins. N-glycosylation starts when the protein is co-translationally imported into the Endoplasmic Reticulum (ER) leading to mannose-type N-glycans having from 9 to 5 mannose residues which are highly conserved in eukaryotes. Further processing of N-glycans occurs in the Golgi apparatus through the action of various glycosyltransferases. This step leads to mature N-glycosylated proteins harboring organism-specific complex N-glycan structures. Moreover, for a given glycoprotein, N-glycans are often not homogeneous resulting in a pool of structurally distinct oligosaccharides and therefore various glycoforms of said glycoprotein. Complex glycans and heterogeneity can constitute drawbacks of currently available expression systems for the production of recombinant glycoproteins. The occurrence of various glycoforms can thus compromise batch-to-batch consistency resulting in quality and regulatory issues in the production of recombinant drugs. Organism-specific complex N-glycans can also result in issues when using non-human cells. For example, glycoproteins produced in CHO cells can contain N-glycolylneuraminic acid whereas those produced in murine cells contain galactose-α(1,3)-galactose which are respectively potentially and highly immunogenic for human. Similarly, insect- or plant-based expression systems also contain potentially immunogenic epitopes such as α(1,3)-fucose (in insect and plant cells) and β(1,2)-xylose (in plant cells).
The present invention discloses the production of glycoproteins and their storage in the plastid of transformed microalgae from the groups of heterokontophytes, haptophytes or cryptophytes. These groups are unique amongst other microalgae or even photosynthetic eukaryotic organisms as they contain plastids surrounded by four membranes with the outermost membrane being interconnected with the ER membrane and studded with ribosomes. This outermost membrane is commonly named Chloroplast Endoplasmic Reticulum (CER). A pathway has been characterized for the targeting of nuclear-encoded proteins in the plastid of these microalgae. Precursors of those proteins are synthesized with an amino-terminal bipartite targeting signal sequence also called "bipartite topogenic signal" (BTS) sequence composed of a signal peptide followed by a transit peptide preceding the sequence of the mature protein. The first step of trafficking in these microorganisms involves the co-translational transport into the ER lumen via the signal peptide. The mechanism for crossing through the second outermost membrane has not been fully identified. Passage through the two innermost membranes to reach the stroma likely involves the transit peptide and translocators showing similarity with plants (see Bolte et al. (2009) Protein targeting into secondary plastids, Journal of Eukaryotic Microbiology, n°56, pp:9-15 for a review of plastids targeting). The evidences of a transport of nuclear-encoded glycosylated proteins to the plastid are scarce and concern plant plastids surrounded by two membranes. These glycoproteins contain complex N-glycan patterns such as β(1,2)-xylose and α(1,3)-fucose residues typical of those added in the Golgi apparatus. The existing literature does not reveal any data concerning the transport of nuclear-encoded glycoproteins to microalgal plastids surrounded by a CER membrane. Besides, background art does not teach or suggest any method for the production of glycoprotein and their storage in the plastidial compartment.
The inventors have surprisingly discovered that the use of a bipartite topogenic signal sequence in a transformed microalga having a CER enabled the expression and glycosylation of proteins in the Endoplasmic Reticulum followed by a transport into the plastid of said microalga without any passage through the Golgi apparatus. N-linked glycans of these glycoproteins are high mannose oligosaccharides (Man-5 to Man-9) characteristic of the ER glycosylation pattern and consequently do not present immunogenic patterns of glycosylation such as those added by glycosyl transferases into the Golgi apparatus. Therefore, the present invention offers an effective method for the production of therapeutic recombinant proteins requiring high mannose glycans for their biological activity without having to inactivate the glycosylation pathway in the Golgi apparatus. Proteins harboring a "high mannose" pattern of glycosylation hold a strong therapeutic interest in the treatment of various diseases. For example, recombinant lysosomal enzymes used for the treatment of lysosomal storage disorders such as Gaucher's or Fabry's diseases require terminal mannose residues for their uptake by human cells. Such glycosylation pattern cannot be directly obtained by CHO cells used for the commercial production of current enzyme replacement therapies and enzymes produced by this system need further deglycosylation steps. The present invention has also applications for the production of viral envelope proteins. For example, the native glycoprotein gp120 of the HIV envelope spike bears N-linked glycans which are almost entirely oligomannose (Man_5-9GlcNAc₂). These glycans of gp120 (Man_6-9GlcNAc₂) are important determinant of antibodies recognition including 2G12, one of the most effective HIV neutralizing antibody. In the context of the viral vaccination design, the present invention thus confers a major advantage over conventional platform such as human cell lines for the production of the envelope glycoproteins bearing high mannose glycans, and used as antigens.
The glycans' homogeneity as opposed to mixture of complex glycan structures obtained in other expression systems also constitutes a major benefit of the present invention in term of product quality and consistency. Furthermore, said method is also effective for the production of a high amount of proteins in a stable environment as said proteins are transported and stored into the plastidial stroma.

SUMMARY OF THE INVENTION

The present invention describes a microalgal-based expression system for the production of glycoproteins and their storage in the plastid. Microalgae used for this invention are species from the groups of heterokonts, cryptophytes and haptophytes that harbor plastid surrounded by an outermost membrane continuous with the Endoplasmic Reticulum. Glycoproteins expressed by mean of the present invention contain targeting sequence allowing their co-translational import into the ER where they undergo N-glycosylation prior to their transport into the plastidial stroma. The present invention enables the production of glycoproteins having a N-glycosylation pattern composed of "high mannose" oligosaccharides, preferably said N-glycosylation pattern being non-immunogenic.

BRIEF DESCRIPTION OF DRAWINGS

DETAILED DESCRIPTION OF THE INVENTION

The invention aims to provide a new system for producing proteins harboring a "high mannose" pattern of glycosylation, said proteins being expressed and glycosylated in the Endoplasmic Reticulum of microalgae and further transported in the plastid of said microalgae without any passage through the Golgi apparatus.
An object of the invention is the use of a transformed microalga for the production of a protein harboring a "high mannose" pattern of glycosylation in the plastid of said transformed microalga, wherein

1) said transformed microalga has a Chloroplast Endoplasmic Reticulum (CER);
2) said microalga is transformed with a nucleic acid sequence operatively linked to a promoter, said nucleic acid sequence encoding an amino acid sequence comprising:
1. (i) An amino-terminal bipartite topogenic signal (BTS) sequence composed of at least a signal peptide followed by a transit peptide ; and
2. (ii) The sequence of said protein.
3) the xylosyltransferases and fucosyltransferases of said microalga have not been inactivated;
4) The N-acetylglycosyltransferase I of said microalga has not been inactivated, preferably the N-acetylglycosyltranferases II, III, IV, V and VI, mannosidase II and glycosyltransferases of said microalga have not been inactivated;

The terms "Chloroplast Endoplasmic Reticulum" or "CER" used herein refer to the outermost membrane of the plastid that is continuous with the Endoplasmic Reticulum membrane to which ribosomes are attached. This CER membrane is only found in plastid interconnected with the Endoplasmic Reticulum and harboring four membranes in heterokonts, cryptophytes and haptophytes. (see Bolte et al. (2009) as disclosed previously, Apt et al. (2002) In vivo characterization of diatom multipartite plastid targeting signals, Journal of Cell Science, n°115, pp:4061-4069).
Therefore, microalgae used herein for the production of a protein harboring a non-immunogenic "high mannose" pattern of glycosylation in the plastid are aquatic photosynthetic microorganisms having a Chloroplast Endoplasmic Reticulum, said microalgae being selected in the group comprising heterokonts, cryptophytes and haptophytes.
In a further embodiment, said microalga of the present invention and having a CER is selected in a group of genus comprising Phaeodactylum, Nitzschia, Skeletonema, Chaetoceros, Odontella, Amphiprora, Thalassiosira, Nannochloropsis, Emiliania, Pavlova, Isochrysis, Apistonema, Rhodomonas.
In a further embodiment of the present invention, the microalga having a CER is the diatom Phaeodactylum tricornutum.
The term "nucleic acid sequence" used herein refers to DNA sequences (e.g., cDNA or genomic or synthetic DNA) and RNA sequences (e. g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. Preferably, said nucleic acid sequence is a DNA sequence. The nucleic acid can be in any topological conformation, such as linear or circular.
"Operatively linked" promoter and terminator refers to a linkage in which the promoter is contiguous with the gene of interest, said gene being also contiguous with the terminator in order to control both the expression and transcriptional termination of said gene.
Examples of promoter that drives expression of a polypeptide in a transformed microalga according to the invention include, but are not restricted to, endogenous nuclear promoters such as the promoter of the fucoxanthin-chlorophyll protein A (pFcpA) (GenBank accession number AF219942 from 1 pb to 142 pb, SEQ ID N°38) and the promoter of the fucoxanthin-chlorophyll protein B gene (pFcpB) (GenBank accession number AF219942 from 848 pb to 1092 pb, SEQ ID N°39) from Phaeodactylum tricornutum and exogenous promoters such as the promoter 35S (pCaMV35S) from the cauliflower mosaic virus (GenBank accession number AF502128 from 25 pb to 859 pb, SEQ ID N°40), the promoter of the Nopaline Synthase (pNOS) from Agrobacterium tumefaciens (GenBank accession number X01077, SEQ ID N°41).
Examples of terminator enabling the transcription termination of a gene in a transformed microalga according to the invention include, but are not restricted to, endogenous nuclear terminators such as the terminator of the fucoxanthin-chlorophyll protein A (tFcpA) (GenBank accession number AF219942 from 1468 pb to 1709 pb, SEQ ID N°42) from Phaeodactylum tricornutum and exogenous terminators such as the terminator of the Nopaline Synthase (tNOS) from Agrobacterium tumefaciens (GenBank accession number AF502128 from 2778 pb to 3030 pb, SEQ ID N°43).
Transformation of microalgae can be carried out by conventional methods such as microparticles bombardment, electroporation, glass beads, polyethylene glycol (PEG), silicon carbide whiskers, or use of viruses or agrobacterium (see León and Fernández (2007) Transgenic microalgae as green cell factories, Landes Bioscience).
In a preferred embodiment, the transformation of a microalga according to the invention is a nuclear transformation for the integration of a foreign nucleic acid sequence into the nuclear genome of said microalga, wherein said nucleic acid sequence may be introduced via a plasmid, virus sequences, double or single strand DNA, circular or linear DNA.
It is generally desirable to include into each nucleotide sequences used for genetic transformation at least one selectable marker to allow selection of microalgae that have been transformed.
Examples of such markers for the transformation of microalga according to the invention are antibiotic resistant genes such as the bleomycin resistance gene (sh ble) enabling resistance to zeocin, nourseothricin resistance genes (nat or sat-1) enabling resistance to nourseothricin, hygromycin phosphotransferase II gene (hptII) enabling resistance to hygromycin or Aminoglycoside-O-phosphotransferase VIII gene (AphVIII) enabling resistance to paromomycin (see also León and Fernández (2007) as disclosed previously).
Alternatively, complementation of auxotrophic mutant strains of microalgae using genes that enable the reversion to prototrophy offers a selection system without the need of antibiotics. Examples of such complementation system include amino acid auxotrophs.
After transformation of microalgae, transformants producing the desired proteins accumulating into the plastidial stroma are selected by the abovementioned selection methods. Alternatively, analysis of the protein to be produced can also be used as a mean of selection on whole microalgae by one or more conventional methods comprising: fluorimeter or immunocytochemistry by exposing cells to an antibody having a specific affinity for the desired protein. This type of selection can also be carried out on disrupted cells by one or more conventional methods comprising: enzyme-linked immunosorbent assay (ELISA), mass spectroscopy such as MALDI-TOF-MS, ESI-MS chromatography or spectrophotometer.
The term "signal peptide" as used herein refers to an amino acid sequence located at the amino terminal end of a polypeptide and which mediates the co-translational transport of said polypeptide across the CER membrane and into the ER lumen where cleavage of the signal peptide finally occurs. This signal peptide is typically 15-30 amino acids long, and presents a 3 domains structure (von Heijne (1990) The signal Peptide, Journal of Membrane Biology, n°115, pp:195-201; Emanuelsson et al. (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nature Protocols, n°2, pp:953-971), which are as follows:

(i) an N-terminal region (n-region) containing positively charged amino acids, such as Arginine (R), Histidine (H) or Lysine (K);
(ii) a central hydrophobic region (h-region) of at least 6 amino acids containing hydrophobic amino acids such as Alanine (A), Cysteine (C), Glycine (G), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Proline (P), Tryptophan (W) or Valine (V); and
(iii) a C-terminal region (c-region) of polar uncharged amino acids such as Asparagine (R), Glutamine (Q), Serine (S), Threonine (T) or Tyrosine (Y). Said C-region often contains a helix-breaking proline or glycine that helps define a cleavage site. Small uncharged residues in positions -3 and -1 (defined as the number of residue before the cleavage site) are usually requires for an efficient cleavage by signal peptidase following the translocation across the endoplasmic reticulum membrane (von Heijne (1990) as disclosed previously; Verner and Schatz (1988) Protein translocation across membranes, Science, n°241, pp:1307-1313).

A person skilled in the art is able to simply identify a signal peptide in an amino acid sequence, for example by using the SignalP 3.0 program (accessible on line at http://www.cbs.dtu.dk/services/SignalP/) which predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms by using two different models: the Neural networks and the Hidden Markov models (Emanuelsson et al. (2007) as disclosed previously).
The term "transit peptide" as used herein refers to an amino acid sequence that is contiguous to the amino terminal end of a polypeptide and which is sufficient to mediate the transport of said polypeptide through the 3 innermost membranes of the plastid and into the stroma of the plastid where the transit peptide is then cleaved off.
Transit peptides show a broad heterogeneity in term of structural features. They vary in length between 8-150 amino acids.
Preferably, the transit peptide according to the invention comprises less than 60 amino acids.
Transit peptides targeting proteins into the plastidial stroma comprise an aromatic residue such as phenylalanine, tryptophan, or tyrosine at position +1 of the transit peptide relative to the signal peptide's predicted cleavage site.
Transit peptides also possess a high content of hydroxylated residues as well as an overall positive charge due to the presence of basic, positively charged amino acids such as lysine, arginine or histidine and a low content of acidic, negatively charged residues such as aspartate and glutamate (Jarvis (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants, New Phytologist, n°179, pp:257-285). The overall charge of said transit peptide can be calculated as the number of basic, positively charged residues minus the number of acidic, negatively charged residues.
In a preferred embodiment, said transit peptide comprises at least 10% of hydroxylated residues, more preferably at least 13% of hydroxylated residues.
In a preferred embodiment, the overall charge of said transit peptide is at least +1, more preferably at least +2.
A person skilled in the art is able to simply identify a transit peptide in an amino acid sequence, for example by using the TargetP program (accessible on line at http://www.cbs.dtu.dk/services/TargetP/) or iPSORT program (accessible on line at http://ipsort.hgc.jp/). Alternatively, the Hectar program can also be used for the prediction of the whole bipartite topogenic signal sequence in heterokonts (accessible on line at http://www.sb-roscoff.fr/hectar/).
The term "bipartite topogenic signal" sequence or "BTS" as used herein refers to an amino acid sequence that is contiguous to the amino terminal end of a polypeptide and composed of a signal peptide adjoining a transit peptide. Said BTS sequence leads to the co-translational import in the Endoplasmic Reticulum further followed by the transport of the protein in the plastid of the microalga, said protein according to the invention harboring a non-immunogenic "high mannose" pattern of glycosylation. The cleavage of the signal peptide of the aforementioned bipartite topogenic signal sequence in the Endoplasmic Reticulum leads to the exposure of the transit peptide for further targeting of the protein to be produced in the plastid of the transformed microalga.
The bipartite topogenic signal sequence can be identified by bioinformatic analyses performed on protein sequences of heterokonts, cryptophytes or haptophytes from publicly available databases such as the US Department of Energy Joint Genome Institute (JGI, http://vvww.jgi.doe.gov/). The putative protein sequences are screened for the presence of signal peptides using SignalP 3.0. Based on the program's prediction cleavage site, retained sequences are processed to remove amino acids corresponding to the signal peptides and further screened for the presence of transit peptide using TargetP or iPSORT. The transit peptides identified are checked for their overall net charge, the content of hydroxylated residues and the occurrence of an aromatic amino acids at position +1 as previously described. Bipartite topogenic signal sequences can then be retrieved by multi-steps analysis and used in-frame for the targeting of proteins to be produced.
In a preferred embodiment, the invention relates to the use of a transformed microalga for the production of a protein harboring a non-immunogenic "high mannose" pattern of glycosylation in the plastid of said transformed microalga, wherein

1) said transformed microalga has a Chloroplast Endoplasmic Reticulum (CER);
2) said microalga is transformed with a nucleic acid sequence operatively linked to a promoter , said nucleic acid sequence encoding an amino acid sequence comprising:
1. (i) An amino-terminal bipartite topogenic signal (BTS) sequence composed of at least a signal peptide followed by a transit peptide ; and
2. (ii) The sequence of said protein.
3) the xylosyltransferases and fucosyltransferases of said microalga have not been inactivated;
4) The N-acetylglycosyltransferase I of said microalga has not been inactivated, preferably the N-acetylglycosyltranferases II, III, IV, V and VI, mannosidase II and glycosyltransferases of said microalga have not been inactivated;
5) Said BTS sequence leads to the co-translational import of the protein into the Endoplasmic Reticulum followed by the transport of said protein in the plastid of the microalga, said protein harboring a "high mannose" pattern of glycosylation.

In another preferred embodiment, the bipartite topogenic signal sequence is selected in the group comprising:

(a) the amino acid sequence set forth in SEQ ID N°1 from the Light Harversting Complex Protein 11 LHCP11 of Guillardia theta;
(b) the amino acid sequence set forth in SEQ ID N°2 from the chloroplast ATPase Gamma subunit (AtpC) protein of P. tricornutum;
(c) the amino acid sequence set forth in SEQ ID N°3 from the triose Phosphate/Phosphate Translocator (Tpt1) protein of P. tricornutum;
(d) the amino acid sequence set forth in SEQ ID N°4 from the Fucoxanthin-chlorophyll a-c binding protein D (FcpD) protein of P. tricornutum;
(e) the amino acid sequence set forth in SEQ ID N°5 from the Fructose-1,6-bisphophatase (FBPC4) protein of P. tricornutum;
(f) the amino acid sequence set forth in SEQ ID N°6 from the Oxygen-evolving Enhancer 1 (OEE1) protein of P. tricornutum;

(a) the amino acid sequence set forth in SEQ ID N°2;
(b) the amino acid sequence set forth in SEQ ID N°3;
(c) the amino acid sequence set forth in SEQ ID N°1;

The term "polypeptide" or "protein" as used herein refers to an amino acid sequence comprising amino acids which are linked by peptide bonds. A polypeptide may be monomeric or polymeric. Furthermore, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.
The term "glycosylated polypeptide/protein" or "glycoprotein" as used herein refers to a protein with N-glycosylation.
The protein to be produced according to the invention is a protein harboring a "high mannose" pattern of glycosylation.
The term "N-glycan" as used herein refers to a N-linked oligosaccharide, e.g., one that is attached by a linkage between the N-acetylglucosamine of said oligosaccharide and an asparagine residue at a site of N-glycosylation.
The term "site of N-glycosylation" refers to the asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, of a protein.
The expressions "high mannose pattern of N-glycosylation" in reference to a protein or "high mannose N-glycosylated protein" refer to a protein harboring high mannose N-linked oligosaccharides, i.e, a protein having on each occupied site of N-glycosylation a glycan composed of 5 to 9 mannose residues and at least one exposed mannose residue (terminal mannose residue)..
Preferably, said protein comprises on each occupied N-glycosylation site a glycan composed of 6 to 9 mannose residues and at least one exposed mannose residue.
The term "occupied site of N-glycosylation" refers to a site of N-glycosylation harboring a N-glycan, i.e. to an asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, harboring oligosaccharides.
Preferably, the protein to be produced according to the invention has at least 5 to 9 mannoses residues, most preferably from 6 to 9 mannose residues on oligosaccharides located at the level of the asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, of said protein.
Preliminary information about N-glycan of the recombinant protein can be obtained by affino- and immunoblotting analysis using specific probes such as lectins (ConA from Canavalia ensiformis; GNL from Galanthus nivalis; HHL from Hippeastrum hybrid ; ECA from Erythrina cristagalli; SNA from Sambucus nigra; MAA from Maackia amurensis ...) and specific N-glycan antibodies (anti-β(1,2) -xylose; anti-α(1,3)-fucose; anti-Neu5Gc, anti-Lewis ...). To investigate the detailed N-glycan profile of recombinant protein, N-linked oligosaccharides is released from the protein in a non specific manner using enzymatic digestion or chemical treatment. The resulting mixture of reducing oligosaccharides can be profiled by HPLC and/or mass spectrometry approaches (ESI-MS-MS and MALDI-TOF essentially). These strategies, coupled to exoglycosidase digestion, enable N-glycan identification and quantification (Dolashka et al. (2010) Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin, Carbohydrate Research, n° 345, pp:2361-2367).
In a preferred embodiment, the protein to be produced according to the invention is a protein harboring a non-immunogenic "high mannose" pattern of glycosylation.
The expression "non-immunogenic pattern of glycosylation" in reference to a protein to be produced according to the invention refers to a pattern of glycosylation which does not elicit an immune response in the human body. As an example, immunogenic pattern of glycosylation comprising β(1,2)-xylose, α(1,3)-fucose, N-glycolylneuraminic acid and/or galactose-α(1,3)-galactose on N-glycans may elicit immune response. Said non-immunogenic pattern of glycosylation of the protein according to the invention arises from the absence of transit of said protein through the Golgi apparatus in which immunogenic patterns are usually added by glycosyltransferases.
Preferably, a non-immunogenic pattern of glycosylation refers to a pattern of glycosylation not harboring any α(1,3)-fucose or β(1,2)-xylose on N-glycans, i.e. on oligosaccharides located at the level of the asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, of said protein.
In a preferred embodiment, the protein produced in the plastid of microalga according to the invention presents a homogenous pattern of glycosylation.
The term "homogenous" refers to a pattern of glycosylation comprising a majority of "high mannose" N-glycans.
Adavantageously, a homogenous pattern of glycosylation comprises at least 70%, preferablyat least 80% or at least 90%, and most preferably at least 95% of "high mannose" N-glycans.
In another most preferred embodiment, the protein presenting a homogenous pattern of glycosylation according to the invention does not comprise galactose, sialic acid, fucose and/or xylose on N-glycans.
The determination of the homogeneity of a pattern of glycosylation can be realized by analyzing N-glycans as described previously.
Preferably, the spectrum of released N-glycans from the protein produced according to the invention does not comprise peaks corresponding to oligosaccharides having at least one of the following sugar residues: galactose, sialic acid, fucose, xylose.
In a preferred embodiment, the protein according to the invention is a heterologous protein.
The term "heterologous", with reference to a protein, means an amino acid sequence which does not exist in the corresponding microalga before its transformation. It is intended that the term encompasses proteins that are encoded by wild-type genes, mutated genes, and/or synthetic genes.
In a still preferred embodiment, said heterologous protein which is produced and transported in the plastid of a transformed microalga according to the invention can be a protein used for therapeutic purposes, wherein said protein can be of viral or animal origin. Preferably, said animal polypeptide is of mammalian origin. Most preferably, said mammalian polypeptide is of human origin.
In a preferred embodiment, the protein according to the invention is a protein selected in the group comprising human lysosomal enzymes, viral envelope glycoproteins or viral envelope glycoprotein's fragments, antibodies or antibody's fragments and derivatives thereof.
The term "lysosomal enzyme" refers to hydrolases that are naturally produced by the human body having an enzymatic activity in the lysosome organelle. Said enzyme is responsible for breaking down complex chemicals, macromolecules or other materials contained in the lysosome. Deficiencies of such enzymes are responsible for the accumulation of lysosomal metabolites leading to pathologies known as lysosomal storage disorders. Examples of such deficient enzymes and related diseases include:

α-fucosidase (Fucosidosis)
α-galactosidase A (Fabry disease)
α-L-iduronidase (Hurler syndrome; Mucopolysaccharidosis type I)
Iduronate-2-sulphatase (Hunter syndrome; Mucopolysaccharidosis type II)
Arylsulfatase B (Maroteaux-Lamy syndrome; Mucopolysaccharidosis type VI)
Acid α-mannosidase (α-mannosidosis)
α-neuraminidase (sialidosis)
Acid α- glucosidase (Pompe disease)
Acid β-galactosidase (GM1 gangliosidosis)
Acid β-glucosidase (Gaucher disease)
β-glucuronidase (Sly syndrome; MPS VII)
Acid β-mannosidase (β-mannosidosis)
Acid Sphingomyelinase (Niemann-Pick disease)
Lysosomal acid lipase (Wolman disease)

Examples of lysosomal enzymes to be produced using the present invention include α-galactosidase A, α-fucosidase, α-L-iduronidase, iduronate-2-sulfatase, arylsulfatase B, acid sphingomyelinase, acid α-mannosidase, acid α-glucosidase, α-neuraminidase, acid β-galactosidase, acid β-glucosidase, β-glucuronidase, acid β-mannosidase and lysosomal acid lipase.
Lysosomal enzymes produced according to the invention harbor high-mannose oligosaccharides and therefore can be taken up by cells through their mannose receptors to replace deficient enzymes in the so-called enzyme replacement therapy (ERT).
The term "Viral envelope glycoprotein" refers to a glycosylated protein included in the viral envelope covering the protein capsid of the virion particle. Said viral envelope glycoprotein is located on the surface of the envelope enabling the binding of the virion particle onto receptors of host cell leading ultimately to entry of the virus into the cell.
Examples of such viral envelope glycoproteins are the precursor gp160 and its processed forms gp120 and gp41 proteins from type 1 human immunodeficiency virus (HIV), E1 and E2 proteins from hepatitis C virus, the E protein from the dengue virus and west nile virus, the GP protein from Ebola virus.
The term "viral envelope glycoprotein's fragments" as used herein refers to fragments of said envelope glycoprotein.
An "antibody" is an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, two identical heavy (H) chains (about 50-70 kDa when full length) and two identical light (L) chains (about 25 kDa when full length) inter-connected by disulfide bonds. Light chains are classified as kappa and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Each heavy chain is comprised of an amino-terminal heavy chain variable region (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains (CH1, CH2, and CH3) for IgG, IgD, and IgA; and 4 domains (CH1, CH2, CH3, andCH4) for IgM and IgE. Each light chain is comprised of an amino-terminal light chain variable region (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The HCVR and LCVR regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with well-known conventions. The functional ability of the antibody to bind a particular antigen depends on the variable regions of each light/heavy chain pair, and is largely determined by the CDRs.
The term "antibody", as used herein, refers to a monoclonal antibody per se. A monoclonal antibody can be a human antibody, chimeric antibody and/or humanized antibody.
Antibodies to be produced according to the invention are for example recombinant IgG antibodies having enhanced antibody dependent cell-mediated cytotoxicity (ADCC).
The term "antibody fragments" as used herein refers to antibody fragments that bind to the particular antigens of said antibody. For example, antibody fragments capable of binding to particular antigens include Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention. Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH₁ domain and/or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
As used herein, the term "derivative" refers to a polypeptide having a percentage of identity of at least 90% with the complete amino acid sequence of any of the protein disclosed previously and having the same activity.
Preferably, a derivative has a percentage of identity of at least 95% with said amino acid sequence, and preferably of at least 99% with said amino acid sequence.
As used herein, "percentage of identity" between two amino acids sequences, means the percentage of identical amino acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences. As used herein, "best alignment" or "optimal alignment", means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences are usually realized by comparing these sequences that have been previously aligned according to the best alignment; this comparison is realized on segments of comparison in order to identify and compare the local regions of similarity. The best sequences alignment to perform comparison can be realized by using computer softwares using algorithms such as GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA. To get the best local alignment, one can preferably used BLAST software, with the BLOSUM 62 matrix, preferably the PAM 30 matrix. The identity percentage between two sequences of amino acids is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.

In a most preferred embodiment of the invention, proteins to be produced according to the invention are selected in the group comprising the sequences disclosed in Table I or derivatives thereof, wherein said protein sequences are fused downstream of a bipartite topogenic signal peptide.

Table I

PROTEIN	CDS SEQ ID N°	Accession number (Protein)	Comments
β-glucocerebrosidase = Acid β-glucosidase	SEQ ID N° 7	AAA35873	Lysosomal enzyme
α-Galactosidase A	SEQ ID N°8	NP_000160	Lysosomal enzyme
Alglucosidase = Acid α-glucosidase	SEQ ID N°9	NP_000143	Lysosomal enzyme
α-L-iduronidase	SEQ ID N°10	NP_000194	Lysosomal enzyme
Iduronate 2-sulfatase	SEQ ID N°11	NP_000193	Lysosomal enzyme
Arylsulfatase B	SEQ ID N°12	NP_000037	Lysosomal enzyme
Acid Sphingomyelinase	SEQ ID N°13	NP_000534	Lysosomal enzyme
Lysosomal acid lipase	SEQ ID N°14	NP_001121077	Lysosomal enzyme
GP120	SEQ ID N°15	NP_579894	Envelope glycoprotein from Human Immunodeficiency Virus 1
GP41	SEQ ID N°16	NP_579895	Envelope transmembrane glycoprotein from Human Immunodeficiency Virus 1
E1 protein	SEQ ID N°17	From aa 192 to 383 of the polyprotein P27958	Envelope glycoprotein from Hepatitis C Virus
E2 protein	SEQ ID N°18	From aa 384 to 746 of the polyprotein P27958	Envelope glycoprotein from Hepatitis C Virus
E protein	SEQ ID N°19	From aa 281 to 775 of the polyprotein AD097105	Envelope glycoprotein from Dengue virus 1
E protein	SEQ ID N°20	From aa 291 to 791 of the polyprotein ADL27981	Envelope glycoprotein from West Nile Virus
Spike glycoprotein precursor	SEQ ID N°21	ACI28632	Envelope glycoprotein from Ebola virus
immunoglobulin heavy chain constant region gamma	SEQ ID N°22	CAC20454	Gamma 1
immunoglobulin heavy chain constant region gamma	SEQ ID N°23	CAC20457	Gamma 4
Immunoglobulin Variable Heavy Chain	SEQ ID N°24	AAA59127
Immunoglobulin Kappa light Chain (VL+CL)	SEQ ID N°25	CAA09181

Still most preferably, said glycosylated protein is the β-glucocerebrosidase as encoded by the amino acid sequence set forth in SEQ ID N°7, the α-Galactosidase A as encoded by the amino acid sequence set forth in SEQ ID N°8, the α-L-iduronidase as encoded by the amino acid sequence set forth in SEQ ID N°10, the Alglucosidase as encoded by the amino acid sequence set forth in SEQ ID N°9, the Acid Sphingomyelinase as encoded by the amino acid sequence set forth in SEQ ID N°13, the GP120 (HIV) as encoded by the amino acid sequence set forth in SEQ ID N°15, the E1 (HCV) as encoded by the amino acid sequence set forth in SEQ ID N°17 and the E2 (HCV) as encoded by the amino acid sequence set forth in SEQ ID N°18.
According to the invention, xylosyltransferases and fucosyltransferases from the microalga used for the production of a protein harboring a non-immunogenic "high mannose" pattern of N-glycosylation have not been inactivated.
The expression "not to have been inactivated" with reference to an enzyme means that the activity of said enzyme in the microalga of the invention has neither been modified nor suppressed by its transformation.
Xylosyltransferases are enzymes having an activity of adding β(1,2)-linked xyloses on N-glycans of glycoproteins in the Golgi apparatus.
Fucosyltransferases are enzymes having an activity of adding α(1,3)-linked fucoses on N-glycans of glycoproteins in the Golgi apparatus.
Still according to the invention, the N-acetylglucosaminyltransferase I has not been inactivated.
The N-acetylglucosaminyltransferase I is capable of adding an N-acetylglucosamine (GlcNac) residue to Man₅GlcNac₂ to produce GlcNacMan₅GlcNac₂ in the Golgi apparatus.
Preferably, the N-acetylglucosaminyltranferases II, III, IV, V and VI, the mannosidase II and the glycosyltransferases of the glycosylation pathway of said microalga have not been inactivated.
Glycosyltransferases comprise galactosyltransferases, fucosyltransferases, xylosyltransferases and sialyltransferases.
Another object of the invention is a vector comprising a nucleic acid sequence operatively linked to a promoter, wherein said nucleic acid sequence encodes an amino acid sequence comprising:

i) a bipartite topogenic signal (BTS) composed of at least a signal peptide and a transit peptide as defined previously, and
ii) the sequence of a protein to be produced as defined previously.

The term "vector" refers to any vehicle capable of facilitating the transfer of a nucleic acid sequence in a microalga. Said term "vector" encompasses without limitation the plasmids, cosmids, phagemids or any other vehicle derived from viral or proteic sources which have been manipulated for the insertion or incorporation of a nucleic acid sequence into a microalga.
In a preferred embodiment, the vector according to the invention also comprises a nucleic acid sequence encoding a selectable marker operatively linked to a promoter as defined previously. Alternatively, a nucleic acid sequence encoding a protein enabling the restoration of prototrophy operatively linked to a promoter can be included in the vector of the present invention.
Another object of the invention is a microalga comprising a nucleic acid sequence operatively linked to a promoter, wherein said nucleic acid sequence encodes an amino acid sequence comprising:

i) a bipartite topogenic signal (BTS) composed of at least a signal peptide and a transit peptide as defined previously, and
ii) the sequence of a protein as defined previously.

Another embodiment of the invention discloses a microalga comprising a vector as defined previously.
Another object of the invention is a method for producing a protein in a transformed microalga having a Chloroplast Endoplasmic Reticulum (CER) as defined previously, said method comprising the steps of:

1) Culturing said transformed microalga;
2) Harvesting the plastid of said transformed microalga;
3) Purifying said protein from said plastid.

The culture of the transformed microalga according to the invention can be carried out by conventional methods of culture according to the specie of the microalga which has been selected for the transformation and production of proteins. A protocol that can be used for the cultivation of microalgae of the present invention is given in the example section.
Method for isolation of plastid from transformed microalga according to the invention includes, but is not limited to, the use of density gradient centrifugation. Said method includes an initial step to release the microalgal cell content by homogenization in a medium containing sorbitol followed by a purification step on a 40% Percoll continuous gradient. Alternatively, the method of "cell disruption" leading to the release of the whole intracellular content can be used for the releasing of the plastid content. A method for cell disruption of microalgae of the present invention by sonication is given in the example section.
The purification of the protein to be produced according to the invention can be carried out by chromatography. Such method includes the use of filtration followed by concanavalin A chromatography to specifically purified glycoproteins. Gel filtration and ion-exchange chromatography can also be used to purify further the recombinant polypeptide.
In another embodiment, the protein of the invention can be fused to an amino- or carboxy-terminal Tag for the purpose of purification of such protein. The term "Tag" as used herein refers to an amino acid sequence fused to a protein. An example of Tag include the histidine tag composed of six histidine residues that can be purified as described in the example section.
In another embodiment, the method for producing a glycoprotein stored in the plastid of said transformed microalga comprises a former step of transforming said microalga with a nucleic acid sequence operatively linked to a promoter as defined previously.
In another embodiment, the method for producing a glycoprotein stored in the plastid of said transformed microalga comprises a former step of transforming said microalga with a vector as defined previously.
In another embodiment of the invention, the method for producing a protein stored in the plastid of a transformed microalga further comprises a step 4) of determining the N-glycosylation pattern of said protein and selecting the protein harboring a "high mannose" pattern of N-glycosylation .
Preliminary information about N-glycosylation of the recombinant protein accumulated in the plastid can be obtained by affinodetection analysis using specific probes such as lectins (ConA from Canavalia ensiformis; GNL from Galanthus nivalis; HHL from Hippeastrum hybrid ; ECA from Erythrina cristagalli; SNA from Sambucus nigra; MAA from Maackia amurensis...). Lack of β(1,2)-xylose, α(1,3)-fucose, Neu5Gc and Lewis epitopes can be assessed by immunoblotting analysis using specific N-glycans antibodies (antiβ(1,2)-xylose; anti-α(1,3)-fucose; anti-Neu5Gc; anti-Lewis ...). To investigate the detailed N-glycan profile of recombinant polypeptide, N-linked oligosaccharides is released from the polypeptide in a non specific manner using enzymatic digestion or chemical treatment. The resulting mixture of reducing oligosaccharides can be profiled by HPLC and/or mass spectrometry approaches (ESI-MS-MS and MALDI-TOF essentially). These strategies, coupled to exoglycosidase digestion, enable N-glycan identification and quantification (Séveno et al. (2008) Plant N-glycan profiling of minute amounts of material, Analytical Biochemistry, n°379, pp:66-72; Dolashka et al. (2010) as disclosed previously).
In a preferred embodiment, the method of producing proteins harboring a "high mannose" N-glycosylation pattern in the plastid of transformed microalgae leads to the transport of at least 70%; preferably 80% and most preferably 90% of said proteins in the plastid.
Said quantity of transported proteins in the stroma of the plastid of said microalgae can be determined using the ratio between the overall quantity of the recombinant protein and the quantity of said protein within the plastid. The overall quantity of recombinant protein is being defined as the sum of intracellular and extracellular quantity of said recombinant protein. Intracellular content of the aforementioned protein can be obtained by cell disruption method while plastidial content of said protein is obtained by purification of said organelle as described previously. Quantities of the recombinant protein can be determined on the intracellular, extracellular and plastidial fractions by enzyme-linked immunosorbent assay (ELISA) on fractions. The percentage of protein stored in the plastid can be calculated as follow: $%_{plastidial} = (Q_{plastidial} x 100) / (Q_{internal} + Q_{external})$
Wherein:

Q_plastidial is the quantity of the recombinant protein to be produced detected in the fraction of purified plastid.
Q_internal is the quantity of the recombinant protein to be produced detected in the intracellular fraction.
Q_external is the quantity of the recombinant protein to be produced detected in the extracellular fraction.
%_plastidial is the percentage of the recombinant protein to be produced accumulated within plastid.

Another object of the invention is a protein harboring a non-immunogenic « high mannose » pattern of glycosylation and produced according to the method as defined previously.
Another object of the invention is a pharmaceutical composition comprising a protein harboring a « high mannose » pattern of glycosylation and produced according to the method of the invention.
In the following examples, the invention is described in more detail with reference to methods. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.

EXAMPLES

Example 1 Targeting of a nuclear-encoded glycosylated chimeric protein into the plastid of Phaeodactylum tricornutum

To test the ability of a nuclear-encoded protein to be glycosylated, targeted and stored into the plastid, Phaeodactylum tricornutum (P. tricornutum) was transformed with a plasmid containing a 54 amino acids bipartite topogenic signal sequence of the phosphoenolpyruvate/phosphate translocator (Tpt1) from P. tricornutum fused in-frame with a sequence coding for a chimeric protein composed of the mature murine erythropoietin (EPO) and the enhanced green fluorescent protein (GFP) (SEQ ID N°26). The chimeric protein is composed of 166 amino acids corresponding to the mature EPO protein lacking its native 26 amino acids signal peptide followed by the PreScission protease cleavage site (LEVLFQGP) and 239 amino acids corresponding to the green fluorescent protein. The chimeric protein obtained contains 3 potential N-glycosylation sites within the EPO sequence.

a) Standard culture conditions of Phaeodactylum tricornutum

The diatom Phaeodactylum tricornutum was grown at 20°C under continuous illumination (280-350 µmol photons.m^-2.s^-1), in natural coastal seawater sterilized by 0.22 µm filtration. This seawater is enriched with nutritive Conway media with addition of silica (40 mg.L^-1 of sodium metasilicate). For large volume (from 2 litters to 300 liters), cultures were aerated with a 2% CO₂/air mixture to maintain the pH in a range of 7.5-8.1.
For genetic transformation, diatoms were spread on gelose containing 1% of agar. After concentration by centrifugation, the diatoms were spread on petri dishes sealed and incubated at 20°C under constant illumination. Concentration of culture was estimated on Mallassez counting cells after fixation of microalgae with a Lugol's solution.

b) Expression constructs for genetic transformation

The cloning vector pPha-T1 (GenBank accession number AF219942) includes sequences of P. tricornutum promoter fcpA (fucoxanthin-chlorophyll a/c-binding proteins A) upstream of a multiple cloning site followed by the terminator fcpA. It also contains a selection cassette with the promoter fcpB (fucoxanthin-chlorophyll a/c-binding proteins B) upstream of the coding sequence sh ble followed by the terminator fcpA (Zaslavskaia and Lippmeier (2000) transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes, Journal of Phycology, n°36,pp379-386). The sequence containing the bipartite topogenic signal sequence fused in-frame with the chimeric EPO-GFP protein (nucleic acid sequence SEQ ID N°26) was synthesized with the addition of EcoRI and HindIII restriction sites flanking the 5' and 3' ends respectively. Alternatively, a similar sequence containing a histidine tag at the carboxy-terminal (EPO-GFP-HisTag) (nucleic acid sequence SEQ ID N°27) was also synthesized with the addition of EcoRI and HindIII restriction sites flanking the 5' and 3' ends respectively. After digestion by EcoRI and HindIII, each insert was introduced into the pPHA-T1 vector. As a control, an empty pPha-T1 vector lacking the EPO-GFP coding sequence was used.

c) Genetic transformation

The transformation was carried out by particles bombardment using the BIORAD PDS-1000/He apparatus modified (Thomas et al. (2001) A helium burst biolistic device adapted to penetrate fragile insect tissues, Jounal of Insect Science, n°1, pp1-9).
Cultures of diatoms (P. tricornutum) in exponential growth phase were concentrated by centrifugation (10 minutes, 2150 g, 20°C), diluted in sterile seawater, and spread on agar plate at 10⁸ cells per plate. The microcarriers are gold particles (diameter 0.6 µm). Microcarriers were prepared according to the protocol of the supplier (BIORAD). Parameters used for shooting were the following:

use of the long nozzle,
use of the stopping ring with the largest hole,
15 cm between the stopping ring and the target (diatom cells),
precipitation of the DNA by a solution containing 1.25 M CaCl₂ and 20 mM spermidine,
a ratio of 1.25 µg DNA for 0.75 mg gold particles per shot,
900 psi rupture disk with a distance of escape of 0.2 cm,
a vacuum of 30 Hg

Diatoms were incubated 24 hours before the addition of the antibiotic zeocin (100 µg.ml^-1) and were then maintained at 20°C under constant illumination. After 1-2 weeks of incubation, individual clones were picked from the plates and inoculated into liquid medium containing zeocin (100 µg.ml^-1).

d) Microalgae DNA extraction

Cells (5.10⁸) transformed by the various vectors were pelleted by centrifugation (2150 g, 15 minutes, 4°C). Microalgae cells were incubated overnight at 4°C with 4 mL of TE NaCl 1X buffer (Tris-HCL 0.1 M, EDTA 0.05 M, NaCl 0.1 M, pH 8). 1% SDS, 1% Sarkosyl and 0.4 mg.mL^-1 of proteinase K were then added to the sample, followed by an incubation at 40°C for 90 minutes. A first phenol-chloroform-isoamyl alcohol extraction was carried out to extract an aqueous phase comprising the nucleic acids. RNA contained in the sample was eliminated by an hour incubation at 60°C in the presence of RNase (1 µg.mL^-1). A second phenol-chloroform extraction was carried out, followed by a precipitation with ethanol. The pellet obtained was air-dried and solubilised into 200 µL of ultrapure sterile water. Quantification of DNA was carried out by spectrophotometry (260 nm) and analysed by agarose gel electrophoresis.

e) Polymerase chain reaction (PCR) analysis

The incorporation of the heterologous chimeric EPO-GFP sequence in the genome of Phaeodactylum tricornutum was assessed by PCR analysis. The sequences of primers used for the PCR amplification were 5'-GTCTATATGAAGCTGAAGGG-3' (SEQ ID N°28) and 5'-GTGAGCAAGGGCGAGGAGC-3' (SEQ ID N°29) located in the EPO and GFP sequence respectively. The PCR reaction was carried out in a final volume of 50 µl consisting of 1X PCR buffer, 0.2 mM of each dNTP, 5 µM of each primer, 20 ng of template DNA and 1.25 U of Taq DNA polymerase (Taq DNA polymerase, ROCHE). Thirty cycles were performed for the amplification of template DNA. Initial denaturation was performed at 94°C for 4 min. Each subsequent cycle consisted of a 94°C (1 min) melting step, a 55°C (1 min) annealing step, and a 72°C (1 min) extension step. Samples obtained after the PCR reaction were run on agarose gel (1%) stained with ethidium bromide.
Results revealed a single band at 276 bp for cells transformed with the constructs carrying the bipartite topogenic signal sequence fused to the chimeric EPO-GFP (data not shown). No band was detected in cells transformed with the control vector. This result validated the incorporation of the exogenous gene in the genome of Phaeodactylum tricornutum.

f) Sub-cellular localization of the chimeric protein

To investigate the sub-cellular localization of the chimeric protein EPO-GFP, confocal microscopy was performed on wild-type and transformed cells of P. tricornutum. GFP and chlorophyll fluorescence were excited at 488 nm, filtered and detected by two different photomultiplier tubes with bandwidths of 500-520 and 625-720 nm for GFP and chlorophyll fluorescence, respectively.
Confocal microscopy revealed the co-localization of the GFP signal (data not shown) with the position of the plastid as observed by bright field and autofluorescence of chlorophyll (data not shown) as well as merged images (data not shown). This result revealed that the use of the amino-terminal bipartite topogenic signal sequence from Tpt1 allowed the targeting of the chimeric protein EPO-GFP to the chloroplast of P. tricornutum.

g) Immunoblotting analysis

Aliquotes of wild-type and transformed cells of P. tricornutum culture at exponential phase of growth are collected and cells are separated from the culture medium by centrifugation (10 minutes, 2150 g, 20°C). Cell pellets are resuspended in Tris-HCl 0.15 M pH 8, saccharose 15%, SDS 0.5%, PMSF 1 mM, protease inhibitor cocktail 1% (SIGMA) and sonicated for 30 min. Cell suspensions obtained are centrifuged (60 minutes, 15000 g, 4°C) to remove cell debris and supernatants correspond to the intracellular fraction.
Ten µL of intracellular fractions from EPO-GFP transformed and wild-type cells are separated by SDS-PAGE using a 12% polyacrylamide gel. The separated proteins are transferred onto nitrocellulose membrane and stained with Ponceau Red in order to control transfer efficiency. The nitrocellulose membrane is blocked overnight in milk 5% dissolved in TBS for immunodetection. Immunodetection is then performed using anti-EPO (R&D SYSTEMS, AF959) (1:500 in TBS-T containing milk 1% for 2h at room temperature) or horseradish peroxidase-conjugated anti-GFP (Santa Cruz, sc-9996-HRP) (1:2000 in TBS-T containing milk 1% for 2h at room temperature). Membrane incubated with the anti-EPO antibody is then washed with TBS-T (6 times, 5 minutes, room temperature) and binding of the primary antibody is revealed upon incubation with a secondary horseradish peroxidase-conjugated rabbit anti-goat IgG (SIGMA-ALDRICH, A8919) (1:10000 in TBS-T containing milk 1% for 1.5h at room temperature). All membranes are then washed with TBS-T (6 times, 5 minutes, room temperature) followed by a final wash with TBS (5 minutes, room temperature). Final development of the blots is performed by chemiluminescence method.Alternatively, deglycosylation assays can be performed on the protein extract prior to immunoblotting experiment using either peptide-N-glycosidase F (PNGase F, New England Biolabs) or endoglycosidase H (Endo H, New England Biolabs) according to manufacturer's recommendations.

h) Purification of the chimeric protein

The chimeric protein EPO-GFP carrying the histidine tag is purified by chromatography method. Intracellular fractions from EPO-GFP-HisTag as well as wild-type cells (control) are prepared as previously described. Both fractions are filtered using a membrane filter of 0.22 µm pore size, concentrated 10 times, and buffer-exchanged with 20 mM Tris, pH 9 containing 5 mM imidazole using a concentration device (MILLIPORE, Amicon Ultra-15, 3 kDa). Purification is performed using the AKTA FPLC system (GE Healthcare) and a Ni Sepharose column (GE Healthcare). The column is equilibrated with 20 mM Tris, pH 9.0 buffer containing 5 mM imidazole and the sample is then loaded. The column is washed with buffer containing 10 mM imidazole followed by elution with buffer containing 200 mM imidazole. The peak is collected and loaded on a Sephadex G-50 column equilibrated with 5 mM sodium phosphate buffer, pH 7.4. The desalted protein is collected, concentrated using a concentration device (MILLIPORE, Amicon Ultra-15, 3 kDa) and analysed by immunoblotting.

i) Structural characterization of N-linked glycans of the chimeric protein

The chimeric EPO-GFP carrying the histidine tag purified by chromatography method is subjected to enzymatic deglycosylation using PNGase F or Endo H in order to release N-linked glycans. Released glycans are analyzed by mass spectrometry as described by Dolashka et al. (2010) Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin, Carbohydr Res, 345:2361-2367.

Example 2 Targeting of nuclear-encoded human lysosomal enzyme into the plastid of Phaeodactylum tricornutum

The β-glucocerebrosidase (GBA) is an enzyme naturally targeted to the lysosomal compartments of human cells. Enzymatic deficiency leads to the accumulation of glucocerebroside in macrophages causing Gaucher's disease. Treatments include enzyme replacement therapy based on the delivery of intravenously injected recombinant β-glucocerebrosidase. The FDA-approved drug is produced in Chinese Hamster Ovary cells and modified by sequential deglycosylation of its carbohydrate side chains to expose alpha-mannosyl residues that mediate uptake of the therapeutic enzyme by surface mannose receptor expressed on target cells. Consequently, there is an industrial benefit to produce a recombinant β-glucocerebrosidase having naturally N-glycans with mannose-terminated structures.
Human β-glucocerebrosidase is expressed, targeted and stored into the plastidial stroma of P. tricornutum by means of the present invention. A plasmid containing a 55 amino acids bipartite topogenic signal sequence of the ATPase gamma subunit (atpC) from P. tricornutum fused in-frame with a 497 amino acids sequence coding for the mature human GBA lacking its native 39 amino acids signal sequence (SEQ ID N°30) is used for the genetic transformation. The GBA protein contains 5 potential N-glycosylation sites.

a) Standard culture conditions of Phaeodactylum tricornutum
Phaeodactylum tricornutum strain used to express GBA is grown and prepared for genetic transformation as in example 1.a).
b) Expression constructs for genetic transformation
The vector used for the expression of human GBA is the same vector used for the expression of the chimeric protein EPO-GFP in example 1.b).
The sequence containing the bipartite topogenic signal sequence fused in-frame with the human GBA (nucleic acid sequence SEQ ID N°30) is synthesized with the addition of EcoRI and HindIII restriction sites flanking the 5' and 3' ends respectively. Alternatively, a similar sequence containing a histidine tag at the carboxy-terminal (GBA-HisTag) is also synthesized (nucleic acid sequence SEQ ID N°31). After digestion by EcoRI and HindIII, each insert is introduced into the pPHA-T1 vector. As a control, an empty pPha-T1 vector lacking the GBA coding sequence is used.
c) Genetic transformation
The genetic transformation carried out in this experiment is described in the previous example 1.c).
d) Microalgae DNA extraction
The DNA extraction carried out in this experiment is described in the previous example 1.d).
e) Polymerase chain reaction (PCR) analysis
The incorporation of the heterologous human GBA sequence in the genome of Phaeodactylum tricornutum is assessed by PCR analysis. The sequences of primers used for the PCR amplification are 5'-ATACCAAGCTCAAGATACC-3' (SEQ ID N°32) and 5'-AACTGTAACTTGTGCTCAGC-3' (SEQ ID N°33) located in the GBA coding sequence. The PCR reaction and agarose electrophoresis of PCR products are carried out as in example 1.e).
f) Immunoblotting analysis
Intracellular fractions of wild-type and transformed cells of P. tricornutum are prepared as previously described in example 1.g).
Ten µL of intracellular fractions from the various GBA expressing cells and wild-type cells are separated by SDS-PAGE using a 12% polyacrylamide gel. The separated proteins are transferred onto nitrocellulose membrane and stained with Ponceau Red in order to control transfer efficiency. Immunoblotting experiment is performed as described in example 1.g) except that the primary antibody is an anti-GBA (Santa Cruz, sc-100544) (1:1000 in TBS-T containing milk 1% for 2h at room temperature) and the secondary antibody is a horseradish peroxidase-conjugated bovine anti-mouse IgG (Santa Cruz, SC2371) (1:10000 in TBS-T containing milk 1% for 1.5h at room temperature).
Deglycosylation assay is performed on the various intracellular fractions as described previously in example 1.g) and analysed by immunoblotting experiment.
g) Purification of the β-glucocerebrosidase
β -glucocerebrosidase carrying the histidine tag (GBA-HisTag) is purified from intracellular fractions by chromatography method as described in example 1.h). Purified β-glucocerebrosidase is then analysed by immunoblotting experiment.
h) Structural characterization of N-linked glycans of the β-glucocerebrosidase
N-linked glycans are released from the β-glucocerebrosidase purified by affinity chromatography and analyzed by mass spectrometry as previously described in example 1.i).

Example 3 Targeting of nuclear-encoded viral envelope glycoprotein into the plastid of Phaeodactylum tricornutum

The envelope spike of HIV contains various highly glycosylated proteins including gp120. Native N-linked glycans of gp120 are almost entirely oligomannose (Man_5-9GlcNAc₂) compared to the recombinant gp120 produced in the human cell line HEK293T which contains a majority of complex glycans. High-mannose glycans of gp120 (Man_6-9GlcNAc₂) are important determinant of antibodies recognition including 2G12, one of the most effective HIV neutralizing antibody. In the context of the viral vaccination design, the present invention thus confers a major advantage for the production of the envelope glycoprotein gp120 bearing high-mannose glycans, and used as antigens.
The viral envelope glycoprotein gp120 is expressed, targeted and stored into the plastidial stroma of P. tricornutum by means of the present invention. A plasmid containing a 55 amino acids bipartite topogenic signal sequence of the ATPase gamma subunit (atpC) from P. tricornutum fused in-frame with a 479 amino acids sequence coding for gp120 (SEQ ID N°34) is used for the genetic transformation. The envelope glycoprotein gp120 contains 24 putative N-glycosylation sites.

a) Standard culture conditions of Phaeodactylum tricornutum
Phaeodactylum tricornutum strain used to express gp120 is grown and prepared for genetic transformation as in example 1.a).
b) Expression constructs for genetic transformation
The vector used for the expression of gp120 is the same vector used for the expression of the chimeric protein EPO-GFP in example 1.b).
The sequence containing the bipartite topogenic signal sequence fused in-frame with gp120 coding sequence containing a stop codon (nucleic acid sequence SEQ ID N°34) is synthesized with the addition of EcoRI and HindIII restriction sites flanking the 5' and 3' ends respectively. Alternatively, a similar sequence containing a histidine tag at the carboxy-terminal (gp120-HisTag) (nucleic acid sequence SEQ ID N°35) is also synthesized with the addition of EcoRI and HindIII restriction sites flanking the 5' and 3' ends respectively. After digestion by EcoRI and HindIII, each insert is introduced into the pPHA-T1 vector. As a control, an empty pPha-T1 vector lacking the gp120 coding sequence is used.
c) Genetic transformation
The genetic transformation carried out in this experiment is described in the previous example 1.c).
d) Microalgae DNA extraction
The DNA extraction carried out in this experiment is described in the previous example 1.d).
e) Polymerase chain reaction (PCR) analysis
The incorporation of the heterologous viral gp120 sequence in the genome of Phaeodactylum tricornutum is assessed by PCR analysis. The sequences of primers used for the PCR amplification are 5'-CACCTCAGTCATTACACAGGC-3' (SEQ ID N°36) and 5'-CCTCCTGAGGATTGCTTAA-3' (SEQ ID N°37) located in the GP120 coding sequence respectively. The PCR reaction and agarose electrophoresis of PCR products are carried out as in example 1.e).
f) Immunoblotting analysis
Intracellular fractions of wild-type and transformed cells of P. tricornutum are prepared as previously described in example 1.g).
Ten µL of intracellular fractions from the various gp120 expressing cells and wild-type cells are separated by SDS-PAGE using a 12% polyacrylamide gel. The separated proteins are transferred onto nitrocellulose membrane and stained with Ponceau Red in order to control transfer efficiency. Immunoblotting experiment is performed as described in example 1.g) with a single anti-histidine tag antibody conjugated to horseradish peroxidase (Santa Cruz, sc-8036-HRP) (1:1000 in TBS-T containing milk 1% for 2h at room temperature).
Deglycosylation assay is performed on the various intracellular fractions as described previously in example 1.g) and analysed by immunoblotting experiment.
g) Purification of the glycoproteins gp120
The glycoprotein gp120 carrying the histidine tag is purified from intracellular fractions by chromatography method as described in example 1.h). Purified gp120 is then analysed by immunoblotting experiment.
h) Structural characterization of N-linked glycan of gp120
N-linked glycans are released from gp120 purified by affinity chromatography and analyzed by mass spectrometry as previously described in example 1.i).

Claims

A transformed microalga producing a protein harboring a non-immunogenic "high mannose" pattern of glycosylation in the plastid of said transformed microalga, wherein
1) said transformed microalga has a Chloroplast Endoplasmic Reticulum (CER);

2) said microalga has been transformed with a nucleic acid sequence operatively linked to a promoter, said nucleic acid sequence encoding an amino acid sequence comprising:
(i) An amino-terminal bipartite topogenic signal (BTS) sequence composed of at least a signal peptide followed by a transit peptide; and

(ii) The sequence of said protein;

3) the xylosyltransferases and fucosyltransferases of said microalga have not been inactivated;

4) The N-acetylglycosyltransferase I of said microalga has not been inactivated, preferably the N-acetylglycosyltranferases II, III, IV, V and VI, mannosidase II and glycosyltransferases of said microalga have not been inactivated.
The transformed microalga according to claim 1 wherein said microalga having a CER is selected from the group comprising heterokonts, cryptophytes and haptophytes microalgae.
The transformed microalga according to any one of claims 1 or 2 wherein said microalga having a CER is selected from the group comprising Phaeodactylum, Nannochloropsis, Nitzschia, Skeletonema, Chaetoceros, Odontella, Amphiprora, Thalassiosira, Emiliania, Pavlova, Isochrysis, Apistonema and Rhodomonas.
The transformed microalga according to any one of claims 1 to 3 wherein said microalga is the diatom Phaeodactylum tricornutum.
The transformed microalga according to any one of claims 1 to 4, wherein said BTS sequence leads to the translocation of the nucleic acid sequence encoding the BTS sequence and the sequence of a protein in the Endoplasmic Reticulum further followed by the transport of said protein in the plastid of the microalga.
The transformed microalga according any one of claims 1 to 5 wherein said protein is a heterologous protein.
The transformed microalga according to any one of claims 1 to 6 wherein said protein present a pattern of glycosylation with at least one exposed mannose residue and between five to nine mannose residues, preferably from six to nine mannose residues, on the oligosaccharides located at the level of the asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, of said protein.
The transformed microalga according any one of claims 1 to 9, wherein said protein is selected in the group comprising lysosomal enzymes, viral envelope glycoproteins, antibodies or antibodies' fragments and derivatives thereof.
The transformed microalga according any one of claims to 1 to 10, wherein said amino acid sequence encoding said protein is selected from the group comprising the amino acid sequences as listed in the following table and derivatives thereof : PROTEIN CDS SEQ ID N° Accession number (Protein) Comments β-glucocerebrosidase = Acid β-glucosidase SEQ ID N° 7 AAA35873 Lysosomal enzyme α-Galactosidase A SEQ ID N°8 NP_000160 Lysosomal enzyme Alglucosidase = Acid α-glucosidase SEQ ID N°9 NP_000143 Lysosomal enzyme α-L-iduronidase SEQ ID N°10 NP_000194 Lysosomal enzyme Iduronate 2-sulfatase SEQ ID N°11 NP_000193 Lysosomal enzyme Arylsulfatase B SEQ ID N°12 NP_000037 Lysosomal enzyme Acid Sphingomyelinase SEQ ID N°13 NP_000534 Lysosomal enzyme Lysosomal acid lipase SEQ ID N°14 NP_001121077 Lysosomal enzyme GP120 SEQ ID N°15 NP_579894 Envelope glycoprotein from Human Immunodeficiency Virus 1 GP41 SEQ ID N°16 NP_579895 Envelope transmembrane glycoprotein from Human Immunodeficiency Virus 1 E1 protein SEQ ID N°17 From aa 192 to 383 of the polyprotein P27958 Envelope glycoprotein from Hepatitis C Virus E2 protein SEQ ID N°18 From aa 384 to 746 of the polyprotein P27958 Envelope glycoprotein from Hepatitis C Virus E protein SEQ ID N°19 From aa 281 to 775 of the polyprotein ADO97105 Envelope glycoprotein from Dengue virus 1 E protein SEQ ID N°20 From aa 291 to 791 of the polyprotein ADL27981 Envelope glycoprotein from West Nile Virus Spike glycoprotein precursor SEQ ID N°21 ACI28632 Envelope glycoprotein from Ebola virus immunoglobulin heavy chain constant region gamma SEQ ID N°22 CAC20454 Gamma 1 SEQ ID N°23 CAC20457 Gamma 4 Immunoglobulin Variable Heavy Chain SEQ ID N°24 AAA59127 Immunoglobulin Kappa light Chain (VL+CL) SEQ ID N°25 CAA09181
A protein harboring a high mannose pattern of glycosylation produced by the method according to any one of claims 12 to 13.
A composition comprising a protein according to claim 10.
A method for producing a protein harboring a "high mannose" pattern of glycosylation in the plastid of a transformed microalga having a Chloroplastic Endoplasmic Reticulum (CER) as claimed in any one of claims 1 to 10, said method comprising the steps of:
1) Culturing said transformed microalga;

2) Harvesting the plastid of said transformed microalga;

3) Purifying said protein from said plastid.
The method according to claim 12 wherein said method comprises a step 4) of determining the glycosylation pattern of said protein and conserving the protein harboring a high mannose pattern of glycosylation.
The use of a transformed microalga having a CER as defined in any one of claims 1 to 9 for the production of a polypeptide harboring a non-immunogenic "high mannose" pattern of glycosylation in the plastid of said microalga.